Sex Differences in Intestinal P-Glycoprotein Expression in Wistar versus Sprague Dawley Rats

Wistar and Sprague Dawley are the most common strains of rat used in pharmaceutical research and are used interchangeably in pre-clinical drug development. No studies have assessed whether Wistar and Sprague Dawley rats are equivalent in the gastrointestinal factors that influence oral drug absorption, specifically in relation to intestinal transporters. Enzyme-linked immunosorbent assay (ELISA) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) are two reliable methods for quantifying intestinal protein levels with their own distinct advantages and limitations. In this study, P-glycoprotein (P-gp), a key efflux transporter, was quantified using ELISA and LC-MS/MS along the complete intestinal tract of male and female Wistar and Sprague Dawley rats. This work presents that Sprague Dawley rats have innately higher baseline P-gp expression than Wistar rats. Significant sex differences in P-gp expression were identified in the jejunum, ileum and colon between male and female Wistar rats using both techniques, with males exhibiting higher P-gp levels. Sprague Dawley rats showed no sex differences in P-gp expression through ELISA and LC-MS/MS. Both methods demonstrated similar trends for P-gp quantification, but ELISA could offer faster data acquisition. Our findings report significant sex differences between the strains and highlight that Wistar and Sprague Dawley rats are not equivalent in their P-gp expression. As humans exhibit distinct sex differences in intestinal P-gp levels, Wistar rats may therefore be a more suitable pre-clinical animal strain to model oral drug absorption of P-gp substrates in male and female subjects.

Introduction
Animal models are an integral pillar in pre-clinical development that have aligned our understanding of the pathologies of diseases and drug-target identification. Rodents have been historically and favourably used across pharmaceutical research to infer clinical decisions considering safety, pharmacokinetics, pharmacodynamics and biodistribution [1,2]. Rats are chosen as model organisms due to their widespread availability, low breeding costs, short reproductive cycle and, to some respect, similarities to humans [3]. The Rattus genus is an umbrella for multiple species of rats. The first documented rat was the wild brown Rattus norvegicus named by English scientist Berkenhout in 1772 who believed that these rats migrated to England from Norwegian ships in 1728 [4]. Over time, rats were bred for sport producing variants including the albino mutant Rattus norvegicus. In 1906, the Wistar Institute bred a strain from the albino mutant Rattus norvegicus, named the Wistar rat [5]. Notably, it was the first standardised rat model to be used in biological and fluoride (PMSF), 1% Nonidet P40 and protease inhibitor cocktail from Sigma (Darmstadt, Germany) in a phosphate-buffered saline (PBS) solution and stored at 4 • C.
The surrogate peptide for P-gp quantification and its stable isotope-labelled internal standard were of analytical grade (purity >95%) and were synthesised and quantified via the amino acid analysis using an Allpeptide (Hangzhou, China). All other chemicals are mentioned individually in the following methods. Rat P-gp ELISA kits (MM-0604R2) were purchased from MEIMAN Biotech (Guangzhou, China).

Animals
In total, 8 male and 8 female Wistar and Sprague Dawley rats (healthy, from 8 to 13 weeks old), respectively, were used as the animal models. The rats were housed at room temperature (25 • C) in a light-dark cycle of 12 h. The rats acclimatised to the animal unit for at least 7 days. On the day before the experiments, the rats were fasted overnight and housed individually in metabolic cages until the following morning at 8 a.m. The protocols were agreed by the Administrative Committee on Animal Research in Sun Yat-sen University (Protocol No. SYSU-20200420; date of approval: 1 January 2021). Full compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines was practiced and followed.

Intestinal Tissue Collection
Their intestines were immediately excised and stored in an ice-cold KBR solution, following sacrifice by CO 2 asphyxiation. The intestines were cut into four segments, roughly 2 cm pieces: duodenum (1 cm from the ligament of Treitz), jejunum (10 cm from the ligament of Treitz), ileum (1 cm from the caecum) and colon (descending), were isolated and opened along their mesenteric border. The tissues were gently washed with KBR solution to remove the intestinal contents.

Initial Total Protein Quantification
The mucosal tissues (approximately 50 mg) were cut into small pieces and homogenized in 0.5 mL of RIPA lysis buffer at 30 Hz for 30 s with a TissueLyser (QIAGEN, Hilden, Germany); this was repeated twice at intervals of 30 s for complete homogenisation. The tissue homogenates were incubated at 4 • C for 2 h, then centrifuged at 12,000× g for 5 min. The total tissue protein was collected in the supernatants; its concentration was subsequently determined with a Pierce™ BCA protein assay kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. To measure the target P-gp transporter protein level by ELISA, ELISA kits (Meimian Biotech, Guangzhou, China) were used based on the manufacturer's description; a volume of the supernatant (from Section 2.4) containing a mass of 50 µg of total protein lysate was taken.
To measure the target P-gp transporter protein level, LC-MS/MS was utilised following a published method [18]. Briefly, a volume of the supernatant (from Section 2.4) containing a mass of 50 µg of total protein lysate was taken and made up to 200 µL with 50 mM ammonium bicarbonate buffer. Dithiothreitol, which was freshly prepared (4 µL; 20 mM), was then added and gently mixed to form the total protein solution. The solution was incubated for 20 min at 56 • C for protein denaturation to occur. After cooling, alkylation was conducted by adding 8 µL of iodoacetamide (375 mM) and was then incubated for 20 min at 37 • C in a dark environment. The precipitation step was carried out by adding 600 µL of cold methanol and 150 µL of cold chloroform (both at 4 • C) to the sample solution. The tube was inverted several times. A volume of 450 µL of cold water was added; then, the sample was immediately centrifuged at 15,000 rpm for 5 min at 4 • C. After centrifugation, the lower layer (below the suspended protein pellet) was removed before the upper layer (above the suspended protein pellet), and an additional 450 µL of cold methanol was added. The tube was inverted to wash the protein pellet. The sample was then centrifuged at 15,000 rpm for 5 min at 4 • C. Immediately after centrifugation, the supernatant was fully removed. Ammonium bicarbonate buffer (47 µL; 50 mM) was added to the precipitated protein pellet. The protein solution was then sonicated for 30 s for a maximum of three cycles until a homogeneous protein suspension was achieved. Human serum albumin was processed under the same conditions for subsequent use as a matrix in the construction of the calibration curve. Trypsin solution (5 µL; 0.5 µg/µL) was added to the re-suspended protein solution and incubated for 4 h at 37 • C. Stable isotope-labelled internal P-gp standard (5 µL; 200 fmol/µL) was added to each sample, and unlabelled peptides were spiked into the human serum albumin matrix to serve as calibration standards or quality control samples toward method validation. The digestion process was stopped by adding 3 µL of 50% formic acid in water. The final processed sample solution (60 µL) was then centrifuged at 15,000 rpm for 5 min at 4 • C, and 30 µL of the supernatant was then obtained for LC-MS/MS analysis. All sample digestion procedures were processed in Protein LoBind ® tubes (Eppendorf, Hamburg, Germany).
A Shimadzu LCMS-8060 system (Kyoto, Japan) coupled with an ACQUITY UPLC CSH C18 column (2.1 mm × 50 mm, 1.7 µm; Waters, MA, USA) was used for the analyses. The mobile phases were 0.1% formic acid in water and 0.1% formic acid in acetonitrile with a flow rate of 0.5 mL/min. The gradient elution procedure started with 98% solvent A for 5 min, followed by a linear gradient from 98% solvent A to 75% solvent A over 10 min; afterwards, 75% solvent A was held for 1 min, then changed to 55% solvent A for an additional 2 min. Then, solvent A was changed back to the original state (98%) and held for 7 min until the end of the analyses. In accordance with previous studies [24,25], the surrogate peptide sequence as a proxy of P-gp abundance and the three multiple reaction monitoring (MRM) transitions were employed; they are listed in Table 1. The calibration curve was established for each MRM transition and P-gp abundance was calculated from the average value of the three transitions. The mass spectrometer was equipped with an electrospray for ionization and operated in the positive ion mode to monitor the three m/z transitions with a 300 • C source temperature, nebuliser at 45 psi, 11 L/min sheath gas flow, 500 V nozzle voltage, 20 collision energy and 7 cell accelerator voltage. All the chromatograms were assessed with LabSolutions LCMS Ver.5.6 (Shimadzu, Kyoto, Japan). Method validation is described in our previous article [18].

Statistical Analysis
The data were expressed as mean ± standard deviation (S.D.) and tested for normality and homoscedasticity. As the former were true, significant differences among groups were then analysed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc analysis using IBM SPSS Statistics 19 (SPSS Inc., Chicago, IL, USA). A significance value of p < 0.05 was used for all tests. The correlations between ELISA and LC-MS/MS for P-gp expression were assessed by Spearman's rank coefficient. Table 2 shows the expression of intestinal P-gp in male and female Wistar rats quantified by ELISA and LC-MS/MS (n = 8 respectively). The trend in male P-gp expression in both techniques was similar, whereby protein levels increased from the proximal to the distal segments of the small intestinal tract but decreased in the colon. Specifically, male P-gp expression was statistically 1.3-and 1.9-times higher in the ileum than in the duodenum, as quantified by ELISA (duodenum, 9.56 ± 1.25 ng/mg vs. ileum, 12.02 ± 0.92 ng/mg) and LC-MS/MS (duodenum, 5307.20 ± 720.63 ng/mg vs. ileum, 10,258.72 ± 1525.14 ng/mg), respectively (p < 0.05). Female P-gp expression, however, was consistent across the small intestine when quantified by ELISA, ranging from 9.86 to 10.56 ng/mg and presenting no statistical differences (p > 0.05) ( Figure 1). By LC-MS/MS, P-gp expression significantly increased from the duodenum to the ileum by LC-MS/MS (p < 0.05) (Figure 2). At the distal segments, P-gp expression significantly dropped in both male and female Wistar rats from the ileum to the colon. By ELISA, colonic P-gp decreased by 22% in males (p < 0.05) and 21% in females from the ileum (p < 0.05). The decrease in colonic P-gp expression was more pronounced with LC-MS/MS, as protein levels reduced by 33% and 36% in males and females, respectively (p < 0.05) ( Figure 2). Sex differences were detected in the same intestinal regions by ELISA and LC-MS/MS with P-gp expression differing in male and female Wistar rats in the jejunum, ileum and colon (p < 0.05). Figures S1 and S2 show good correlation of P-gp expression when quantified by ELISA and LC-MS/MS in male (R 2 = 0.9726) and female (R 2 = 0.9761) Wistar rats, respectively. Table 2. P-glycoprotein expression in male and female Wistar rats quantified by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Intestinal P-gp in Male Wistar Rats
Quantified by ELISA (ng/mg) (n = 8)  Regional and sex differences in P-gp expression in male and female Wistar rats quantified by ELISA. Data are represented as mean ± S.D., n = 8. The * symbol denotes statistical significance between the sexes in an intestinal region (p < 0.05). Regional differences are statistically significant from the small intestinal segments to the colon (p < 0.05). Figure 1. Regional and sex differences in P-gp expression in male and female Wistar rats quantified by ELISA. Data are represented as mean ± S.D., n = 8. The * symbol denotes statistical significance between the sexes in an intestinal region (p < 0.05). Regional differences are statistically significant from the small intestinal segments to the colon (p < 0.05).  , n = 8. The * symbol denotes statistical significance between the sexes in an intestinal region (p < 0.05). Regional differences are statistically significant from the small intestinal segments to the colon (p < 0.05).

Intestinal P-glycoprotein Quantification in Sprague Dawley Rats
The intestinal P-gp expression in male and female Sprague Dawley rats quantified by ELISA and LC-MS/MS is shown in Table 3. As reported in the Wistar rat, the trend in P-gp expression across the complete intestinal tract in Sprague Dawley rats was similar between ELISA and LC-MS/MS quantification. Figures 3 and 4 show that P-gp expression in Sprague Dawley rats increased along the small intestine and experienced a sharp reduction in the colon. Detected by ELISA, P-gp expression from the duodenum to the ileum increased by 2.0 (duodenum, 10.33 ± 1.25 ng/mg vs. ileum, 21.33 ± 1.10 ng/mg) and 1.9 times (duodenum, 10.38 ± 0.94 ng/mg vs. ileum, 19.80 ± 2.19 ng/mg) in males and females, respectively (p < 0.05) (Figure 3). Regional differences across the small intestine in male and female Sprague Dawley rats, however, were more pronounced when quantified by LC-MS/MS. From the duodenum to the ileum, P-gp significantly increased by 2.  (Figure 4). Similar to Wistar rats, a significant reduction in P-gp expression was identified from the small intestine to the colon. Quantification via ELISA showed that in males and females, colonic P-gp expression reduced by 43% and 36% from the ileum, respectively (p < 0.05). From the ileum, colonic P-gp significantly decreased by 49% and 46% in both male and female Sprague Dawley rats when quantified by LC-MS/MS (p < 0.05). Neither the ELISA nor the LC-MS/MS quantification strategy identified any sex differences in P-gp expression across the segments of the intestinal tract of Sprague Dawley rats (Figure 4). Figures S3 and S4 show good correlation of P-gp expression when quantified by ELISA and LC-MS/MS in male (R 2 = 0.9862) and female (R 2 = 0.9760) Sprague Dawley rats, respectively. , n = 8. The * symbol denotes statistical significance between the sexes in an intestinal region (p < 0.05). Regional differences are statistically significant from the small intestinal segments to the colon (p < 0.05).

Intestinal P-glycoprotein Quantification in Sprague Dawley Rats
The intestinal P-gp expression in male and female Sprague Dawley rats quantified by ELISA and LC-MS/MS is shown in Table 3. As reported in the Wistar rat, the trend in P-gp expression across the complete intestinal tract in Sprague Dawley rats was similar between ELISA and LC-MS/MS quantification. Figures 3 and 4 show that P-gp expression in Sprague Dawley rats increased along the small intestine and experienced a sharp reduction in the colon. Detected by ELISA, P-gp expression from the duodenum to the ileum increased by 2.0 (duodenum, 10.33 ± 1.25 ng/mg vs. ileum, 21.33 ± 1.10 ng/mg) and 1.9 times (duodenum, 10.38 ± 0.94 ng/mg vs. ileum, 19.80 ± 2.19 ng/mg) in males and females, respectively (p < 0.05) (Figure 3). Regional differences across the small intestine in male and female Sprague Dawley rats, however, were more pronounced when quantified by LC-MS/MS. From the duodenum to the ileum, P-gp significantly increased by 2.5 times (duodenum, 6633.03 ± 1452. 23 (Figure 4). Similar to Wistar rats, a significant reduction in P-gp expression was identified from the small intestine to the colon. Quantification via ELISA showed that in males and females, colonic P-gp expression reduced by 43% and 36% from the ileum, respectively (p < 0.05). From the ileum, colonic P-gp significantly decreased by 49% and 46% in both male and female Sprague Dawley rats when quantified by LC-MS/MS (p < 0.05). Neither the ELISA nor the LC-MS/MS quantification strategy identified any sex differences in P-gp expression across the segments of the intestinal tract of Sprague Dawley rats (Figure 4). Figures S3 and S4 show good correlation of P-gp expression when quantified by ELISA and LC-MS/MS in male (R 2 = 0.9862) and female (R 2 = 0.9760) Sprague Dawley rats, respectively. Table 3. P-glycoprotein expression in male and female Sprague Dawley rats quantified by enzymelinked immunosorbent assay (ELISA) and liquid chromatography-tandem Mass spectrometry (LC-MS/MS).

Strain Differences in Intestinal P-glycoprotein between Wistar and Sprague Dawley Rats
There were distinct differences in P-gp expression between the Wistar and Sprague Dawley rats (Figures S5-S8). Both ELISA and LC-MS/MS confirmed that P-gp expression was innately higher Sprague Dawley rats in comparison to Wistar types. Specifically, Pgp in the male and female jejunum and ileum, and in the female colon was higher in Spra-

Strain Differences in Intestinal P-glycoprotein between Wistar and Sprague Dawley Rats
There were distinct differences in P-gp expression between the Wistar and Sprague Dawley rats (Figures S5-S8). Both ELISA and LC-MS/MS confirmed that P-gp expression was innately higher Sprague Dawley rats in comparison to Wistar types. Specifically, P-gp in the male and female jejunum and ileum, and in the female colon was higher in Sprague Dawley than Wistar rats by LC-MS/MS (p < 0.05) (Tables 2 and 3). The quantification of protein expression via ELISA, however, was not able to detect strain differences between male Wistar and Sprague Dawley rats (p > 0.05).

Discussion
Wistar and Sprague Dawley rats are the most routinely used strains in pre-clinical drug-absorption studies [8,26]. A notable commentary by Clause, however, stated that by its uniformity, quality, efficiency of production and application to scientific practice, the Wistar rat is the right choice toward a standardised animal model for translation to humans [5].
Researchers select the rat strain for their experiments based on biological features, convention, or even cost. There are, however, key differences in their behaviour, appearance and, importantly, innate physiology. For example, Sprague Dawley rats grow faster than Wistar rats. Wistar rats, on the other hand, show higher survival rates [27]. A study by Rybnikova et al. demonstrated that Sprague Dawley rat strains exhibited higher testosterone levels in comparison to Wistar rats, contributing to their higher reproductive potential [28]. Sprague Dawley rats also presented increased motor activity and explorative levels in comparison to the Wistar rat; however, grooming patterns and depressive-like behaviours were higher in the latter [28].
Our study identified that Sprague Dawley rats had intrinsically higher expression of P-gp (Figures 3 and 4) than Wistar rats (Figures 1 and 2) following quantification by both ELISA and LC-MS/MS. A study by Oltra-Noguera et al., however, found that the mean permeability (P eff ) of verapamil, a known P-gp substrate, was higher in Sprague Dawley rats than in Wistar rats in an in situ closed-loop perfusion model, which implies higher P-gp expression in Wistar rats [29]. On the other hand, a study by Liu et al., reported that P-gpmediated efflux was the limiting factor for intestinal absorption of SNX-2112, a promising anti-cancer agent, in intestinal microsomes from male Sprague Dawley rats [30].
Biological sex can contribute towards differences in physiology, which can in turn influence drug response [31]. In pre-clinical studies, male animals can be preferentially selected over female animals due to the lack of hormonal fluctuations, ease in handling and faster clearance [32,33]. In some cases, male and female animals are pooled together to reduce the number of animals used in line with the 3Rs initiative [34]. However, these strategies ignore sex-related differences in drug performance, with implications for the clinical safety and efficacy of pharmaceutical formulations. For example, the oral bioavailability of P-gp drug substrate ranitidine is markedly higher in male rats when co-formulated with polyoxyethylated excipients, including polyethylene glycol (PEG) 400 [35,36], PEG 2000, Cremophor RH 40, Poloxamer 188 and Tween 80. No differences in ranitidine oral bioavailability, however, was seen in female rats [37]. The implications of PEG 400 were demonstrated to have clinical consequences in humans. Male volunteers showed statistically higher oral bioavailability of the P-gp drug substrates ranitidine [38] and cimetidine [39] than the female counterparts due to the excipient modulating baseline P-gp expression in males alone.
Our results demonstrated distinct differences between the two rat strains, with respect to sex as a biological variable. Small intestinal P-gp in male Wistar rats were statistically higher than their female counterparts when quantified by both ELISA and LC-MS/MS. In contrast, there were no sex differences in P-gp expression seen in Sprague Dawley rats. Oltra-Noguera et al. reported a significant sex difference in the intestinal permeation of verapamil in Wistar rats but not Sprague Dawley rats [29], which corroborates the findings of this study. Evidence of lower P-gp activity in female Wistar rats has been demonstrated by Mariana et al. Rhodamine 123, a well-known P-gp substrate, showed 1.78-fold greater serosal-to-mucosal transport in males than females, which suggests increased P-gp activity [40]. Using a closed-loop model, Yin et al. showed reduced intestinal absorption of P-gp substrate ampicillin in female Wistar rats in comparison with the male counterparts [41].
The optimal site for oral drug absorption in the gastrointestinal tract is dependent on the properties of the drug products and regional differences in the mucus layer, intercellular tight junctions, surface area and the expression of transporters and enzymes in the intestinal epithelial cells [42]. The trend in the P-gp level across the small intestine reflects our groups' previous findings in Western blot and LC-MS/MS, where P-gp increases from the proximal to the distal small intestine and then decreases in the colon [18].
A reliable and fast analytical method is required to support clinical drug development. LC-MS/MS and immunosorbent assays are often utilised to aid the understanding of the effect of pharmaceutical formulations. One application is the quantification of P-gp drug expression to infer for potential interactions or inhibition [18,43,44]. Protein precipitation, conventional liquid/liquid extraction and solid-phase extraction procedures are routine stages of sample preparation for LC-MS/MS. Most of these preparation techniques take at least 1 hour to perform, without the consideration of incubation periods [45]. Although solid phase extraction is relatively simple, there is a drying process to ensure the separation of the protein pellet. This involves the extraction of the solvent; however, if the solvent is not completely removed, issues with reversed-phase liquid chromatography compatibility may arise [46]. Slow and technically difficult sample preparation and long sample cycle times can therefore be a limiting factor in analyses and drug-development decision making. With the demand of fast-paced development and improved toxicological and efficacy understanding for new modalities, high-throughput strategies are being adopted more and more in pharmaceutical research [43]. The 96-well plate is the most used format for bioanalytical sample preparation. Singleplex ELISA assays have been designed to be highly sensitive and quantitative for the detection of target analytes within heterogenous samples [44], with quantification times reduced by at least half in comparison to LC-MS/MS [47]. The work herein identified that the trend in P-gp expression between the sexes and between Wistar and Sprague Dawley rats along the intestinal tract were similar when quantified by ELISA and LC-MS/MS. Sex differences in small intestinal and colonic P-gp levels between ELISA and LC-MS/MS were also appreciated. The significant increase in Pgp expression in female Wistar rats, however, was detected only by LC-MS/MS, potentially due to a wider assay range or immunoaffinity [48]. This finding was less pronounced through the immunosorbent assay method, which may have been due reduced detection sensitivity, restrictive analytical range [49] or the small sample size (n = 8) (Figures 1 and 2). Although both techniques showed similar trends of intestinal expression and displayed good positive correlation (R 2 = 0.9726 -0.9862; Supplementary Figures S1-S4), LC-MS/MS measurements were markedly higher than ELISA measurements due to superior specificity to total protein quantification and invariance to cross-reactions, in comparison to the ELISA method which measures the bound target protein [50,51].
As P-gp expression is higher in Sprague Dawley rats, should this strain be the preclinical animal model of choice for a new modality that is investigated to be a P-gp substrate? Drug response may present decreased oral bioavailability from greater P-gp efflux in comparison to Wistar rats. Higher expression of P-gp may be linked to greater protection from xenobiotics entering systemic circulation [52]. However, the sex differences in P-gp expression as shown in humans [18] are not exhibited in Sprague Dawley rats. Drug development has taken a 'one size fits all' approach towards pharmaceutical manufacturing. The adoption of sex-informed perspectives across the pharmaceutical pipeline, however, increases scientific rigour and encourages novel research [53]. This inclusion in pre-clinical development, therefore, can expedite translation into first-in-human trials if sex is considered as a biological variable in animal studies. Wistar rats demonstrate key sex differences in innate P-gp expression in the small intestine where the majority of immediate release drugs are absorbed. As such, this study advocates for this animal model to be used when investigating potential P-gp drug substrates, so that sex-related influences are powered in research and development [31,54].

Conclusions
This study is the first to identify strain differences in the intestinal P-gp expression between Wistar and Sprague Dawley rats. Wistar rats showed significant sex differences in the P-gp expression along the intestinal tract-duodenum, jejunum, ileum and colon. Sprague Dawley rats, however, presented no sex differences in intestinal P-gp levels. Importantly, both quantification strategies-ELISA and LC-MS/MS-produced similar trends in results concerning intestinal P-gp expression. Although LC-MS/MS may be more sensitive, ELISA can lead to faster data acquisition to effectively guide the next stages of drug development. This study proposes that Wistar rats should be the animal model of choice for pre-clinical investigations concerning the oral development of P-gp drug substrates due to the appreciation of innate sex differences as seen in humans.


Pharmacological inhibition of sphingolipid synthesis reduces ferroptosis by stimulating the HIF-1 pathway

Summary Ferroptosis is crucial to the pathology of many neurological diseases. Here, we found pre-treatment with myriocin, an inhibitor of de novo synthesis of sphingolipid, significantly decreased the erastin- or glutamate-induced ferroptosis of HT22 cells without requiring the recovery of intracellular glutathione. The transcriptome analysis of HT22 cells treated with or without myriocin identified the hypoxia-inducible factor 1 (HIF-1) pathway as a prime and novel drug target. Further study validated that HIF1α was required for the cytoprotective effects of myriocin. Myriocin treatment promoted the expression of HIF-1 pathway effectors including PDK1 and BNIP3 and altered the intracellular levels of glucose metabolites. Additionally, myriocin treatment stabilized HIF1α protein by decreasing its ubiquitination and proteasomal degradation. Similar effects of myriocin on HIF1α stabilization were also found in other mammalian cell lines indicating this is a common mechanism for the cytoprotective role of myriocin.

INTRODUCTION
Oxytosis is a form of regulated cell death (RCD) first described in nerve cells and recently recognized as ferroptosis, which is characterized by iron-dependent, lipid peroxidation, and non-apoptotic cell death (Maher et al., 2020). Ferroptosis has been shown to have significant implications in many neurologic disorders, including stroke (Alim et al., 2019), Alzheimer disease (AD) (Bao et al., 2021), Parkinson disease (PD) (Mahoney-Sanchez et al., 2021), Huntington disease (Mi et al., 2019), and multiple sclerosis (MS) (Hu et al., 2019). While enhanced ferroptosis of cancer cells may benefit patients with cancer, reduced ferroptosis is desirable for the therapies of these neurological diseases. Ferroptosis in neuronal cells can be induced by the glutamate/cystine antiporter, system x c À , inhibitors (erastin or glutamate) (Jiang et al., 2020;Zhang et al., 2020). The inhibition of system x c À results in glutathione depletion, excessive reactive oxygen species (ROS) production, and subsequent ferroptosis. It is important to note that in HT22 cells, N-methyl-d-aspartic-acid receptors (glutamate-gated cation channel) are absent and therefore this cell line is widely used to study glutamate-induced ferroptosis in neuronal cells (Maher and Davis, 1996). Although the in vivo relevance of system x c À inhibition to neurologic disorders is not clear, the in vitro model of system x c À inhibition has been extensively used for screening neuroprotective compounds targeting ferroptosis (Albrecht et al., 2010;Dixon et al., 2014).
Sphingolipids are important building blocks of cellular membranes and play key roles in the maintenance of cellular structural integrity. Additionally, sphingolipids, such as dihydrosphingosine (DHS), ceramides, and sphingosine-1-phosphate, regulate many cellular processes, including proliferation (Samarani et al., 2018), immunity (Seo et al., 2011), aging (Huang et al., 2012), auto/mitophagy (Bekhite et al., 2021), and apoptosis (Giussani et al., 2014). The de novo synthesis of sphingolipid is inhibited by myriocin, which specifically targets serine palmitoyl transferase (SPT) (Wadsworth et al., 2013). Myriocin, also called ISP-1, is a metabolite of the fungus Isaria sinclairii (Miyake et al., 1995). It resembles the transient intermediate formed by SPT in the condensation reaction of L-serine and palmitoyl-CoA (Chen et al., 1999). Our previous studies revealed that myriocin extends the lifespan of yeast cells by enhancing oxidative stress response and regulating homeostasis of intracellular amino acids (Hepowit et al., 2021;Liu et al., 2013). Recent studies have reported that limiting sphingolipid biosynthesis by myriocin had protective effects on many neurological diseases, including AD (Petit et al., 2020), PD (Lin et al., 2018), MS (Dasgupta and Ray, 2019), and Friedreich ataxia (Chen et al., 2016). However, the neuroprotective effect of myriocin against ferroptosis has not been iScience Article addressed. In the present study, we investigated the neuroprotective effect of myriocin on erastin or glutamate-induced ferroptosis in HT22 cells and discovered that the HIF-1 pathway was stimulated by myriocin to attenuate ferroptosis.

Myriocin protects HT22 cells against erastin-or glutamate-induced cell death
The inhibition of system x c À and the resulted oxidative damages of HT22 hippocampal neuronal cells by erastin-or glutamate-induced ferroptosis, which has been implied in the pathology of many neuronal diseases (Bueno et al., 2020;Chu et al., 2020;Neitemeier et al., 2017). To evaluate the potential neuronal cytoprotective effects of myriocin, HT22 cells were pre-treated with 0.5 mM of myriocin for different time from 6 to 48 h followed by treatment with erastin (1 mM) or glutamate (15 mM) for 24 h. The viability of HT22 cells without the pre-treatment of myriocin was significantly decreased by erastin or glutamate, while myriocin pre-treatment for 12 h or more provided significant protection ( Figures 1A and 1B). The protective effect of myriocin is also concentration-dependent from 0.1 to 0.5 mM and the concentrations higher than 0.5 mM are not more beneficial ( Figures 1C and 1D). Additionally, myriocin was able to protect HT22 cells against various concentrations of erastin or glutamate ( Figures 1E and 1F). These data demonstrate that myriocin is an effective compound against erastin-or glutamate-induced death of HT22 cells.
Myriocin is a potent suicide inhibitor of SPT, the first and rate-determining enzyme which produces 3-ketosphinganine in the pathway of the de novo biosynthesis of sphingolipids (Wadsworth et al., 2013). 3-ketosphinganine is then converted to dihydrosphingosine (DHS) by 3-ketosphinganine reductase to produce ceramides and complex sphingolipids ( Figure 1G). In order to validate the hypothesis that myriocin protects HT22 cells against ferroptosis by inhibiting the de novo biosynthesis of sphingolipids, the protective effect of myriocin on the viability of HT22 cells treated with erastin or glutamate was monitored in the presence of DHS. Indeed, the replenishment of DHS reduced the effect of myriocin on cell viability when DHS alone had no impact on cell viability ( Figures 1H and S1). The flow cytometric analysis of dying cells revealed that after 24 h of glutamate or erastin exposure, a major portion of cells was FITC Annexin V positive and PI positive (necrotic or ferroptotic) and a relatively smaller portion of cells was FITC Annexin V positive and PI negative (apoptotic) ( Figure 1I). The pre-treatment of myriocin significantly mitigated the appearance of either apoptotic or necrotic cells, suggesting that myriocin may protect cells from both ferroptosis and apoptosis. And the co-treatment of DHS with myriocin reversed the protective effect of myriocin ( Figure 1I). Therefore, the protective effect of myriocin on cell viability relies on its inhibition of DHS production.

Myriocin decreases erastin-or glutamate-induced oxidative damage independent of GSH
Erastin-and glutamate-induced ferroptosis in neuronal cells is initiated by the inhibition of the Na + -independent cystine/glutamate antiporter, system x c À , which in turn prevents the uptake of cystine and blocks the synthesis of glutathione (Murphy et al., 1989). To investigate if the cytoprotective role of myriocin against erastin-or glutamate-induced cell death is related to the homeostasis of glutathione, we monitored the effect of myriocin on the levels of oxidative and reductive glutathione in HT22 cells. Erastin or glutamate treatment greatly decreased the level of both reduced and oxidized glutathione as expected from previous studies ( Figure 2A) (Herrera et al., 2007;Wang et al., 2020). The pre-treatment of myriocin alone slightly decreased the level of glutathione in cells before erastin or glutamate treatment and it did not recover glutathione levels after erastin or glutamate treatment ( Figure 2A). Therefore, the cytoprotective role of myriocin does not rely on the restoration of GSH level.
Because most of dying cells are ferroptotic ( Figure 1I), we sought to understand how myriocin attenuates erastin-and glutamate-induced ferroptosis. We monitored lipid peroxidation, a hallmark of ferroptosis, and found that while erastin or glutamate treatment significantly enhanced lipid peroxidation 12-fold or 6-fold, respectively, pre-treatment with myriocin significantly suppressed these effects as revealed by (I) Annexin V-FITC/PI flow cytometric analysis of cells pre-treated with or without myriocin (0.5 mM) or DHS (1 mM) for 36 h before incubating with or without erastin (1 mM) or glutamate (15 mM) for 24 h. Histograms show numbers of necrotic and apoptotic cells. For the above, error bars represent the mean G SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

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iScience 25, 104533, July 15, 2022 3 iScience Article flow cytometry using the fluorescent probe C11-BODIPY ( Figure 2B). The effect of myriocin on lipid peroxidation was also inhibited by DHS ( Figure 2B). Additionally, the production of ROS in HT22 cells was analyzed by using a fluorescent probe DCFH-DA. While the pre-treatment of myriocin abolished the surge of ROS production induced by erastin or glutamate treatment, the addition of DHS counteracted the effects of myriocin, and DHS alone had no impact on ROS production ( Figure 2C). Because ferroptosis is iScience Article iron-dependent, the relative labile iron pool (LIP) level in HT22 cells was monitored by the calcein fluorescence quenching method. Both erastin and glutamate treatments increased intracellular LIP in a concentration-dependent manner and the pre-treatment of myriocin significantly attenuated the increase of intracellular LIP induced by erastin or glutamate treatment ( Figures 2D and 2E). These data suggest that inhibiting the biosynthesis of sphingolipids by myriocin protected HT22 cells against erastin-or glutamateinduced ferroptosis by suppressing oxidative damage.

Myriocin activates the HIF-1 signaling pathway
To gain insight into the cytoprotective mechanism of myriocin in HT22 cells, transcriptome profiles of HT22 cells treated with or without 0.5 mM of myriocin were investigated and compared. We identified 113 upregulated and 71 downregulated differentially expressed genes (DEGs) upon myriocin treatment ( Figure S2 and Table S1). Disease and gene correlation network established by homology analysis of DEGs with Homo sapiens genome demonstrates that myriocin treatment regulated the expression of genes involved in metabolic diseases, cardiovascular diseases, cancer, and neurological diseases ( Figure 3A). Gene Ontology (GO) analysis indicates that the downregulated genes were mainly related to cell proliferation and extracellular regulation ( Figure 3B, left part). For upregulated genes, top three GO terms in biological process (BP) enrichment were ''response to hypoxia'', ''positive regulation transcription'', and ''response to cAMP''. The top three GO terms in cell component (CC) enrichment were ''membrane'', ''mitochondrion'', iScience Article and ''transcription factor complex''. And the top three GO terms in molecular function (MF) enrichment were ''transcription factor activity'', ''transcription regulatory region DNA binding'', and ''MAP kinase activity'' ( Figure 3B, right part). These results suggest that myriocin treatment activated the transcription factors related to hypoxia. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs identified 19 signaling pathways significantly altered by myriocin treatment ( Figure 3C). Among them, HIF-1 signaling pathway was the term with the lowest false discovery rate (FDR) second by the term of central carbon metabolism in cancer ( Figure 3C). Note that there was an apparent overlapping of DEGs between these two terms ( Figure 3D). These transcriptome data strongly suggest that myriocin may activate the HIF-1 signaling pathway.
To validate the DEGs regulated by HIF-1 signaling pathway, the expression levels of Eglh3, Bnip3, Glut1, Pfkl, Aldoa, Pdk1, Ldha, and Mct4 were analyzed by qPCR in HT22 cells treated from 6 to 48 h with myriocin. All of these transcripts were increased by myriocin treatment ( Figure 4A). Note that there was only a very minor increase in the mRNA level of Hif1a which is the central regulator of HIF-1 signaling pathway, consistent with the transcriptome analysis (Table S1). However, the immunofluorescence images indicate that the protein level of HIF1a was remarkably elevated by myriocin treatment (Figure 4B), suggesting that it was regulated post-transcriptionally by myriocin. Western blots also reveal that the level of HIF1a protein, together with PDK1 and BNIP3 proteins were significantly increased over the time in myriocin-treated cells ( Figures 4C and S3). Furthermore, this increase in protein levels was prevented by supplementing DHS (Figures 4B and S4).
PDK1 and BNIP3 are among the mostly studied proteins controlled by HIF-1 and play crucial roles in regulating the catabolism of glucose and mitochondrial autophagy, respectively ( Figure 4D) (Kim et al., 2006;Zhang et al., 2008). Therefore, we first asked if the catabolism of glucose was altered by myriocin. Indeed, myriocin treatment inhibited the carbon flux from glycolysis to tricarboxylic acid (TCA) cycle in HT22 cells as revealed by the elevation of intracellular levels of pyruvate and lactate and the reduction of citrate when intracellular glucose was constant ( Figure 4E). This phenomenon resembled the effects of PDK1 upregulation. Additionally, myriocin treatment decreased the content of mitochondrial ( Figure 4F) and increased LC3II, a marker of autophagy ( Figure S5). These changes are consistent to the effects of BNIP3 upregulation. These effects of myriocin were also inhibited by the addition of DHS ( Figures 4E and 4F). Together, these data indicate that myriocin induced the activation of HIF1a and its downstream signals by inhibiting the de novo synthesis of sphingolipids.

The cytoprotective effects of myriocin require HIF1a
To investigate the role of HIF1a in the cytoprotective effects of myriocin, Hif1a was knocked down in HT22 cells. Hif1a knockdown resulted in a drastic decrease of its mRNA and protein levels ( Figures 5A, 5B, and S6), and suppressed the expression of HIF-1 effectors including Pdk1, Bnip3, Glut1, Mct4, and Pfkl which were stimulated by myriocin treatment ( Figure 5A). Additionally, the expression of PDK1 and the intracellular levels of glucose, pyruvate, lactate, and citrate did not respond to myriocin treatment anymore after Hif1a knockdown ( Figures 5A, 5B, 5C and S6), indicating that the regulation of PDK1 and glycolysis by myriocin requires HIF1a. Similarly, consistent with the expression of Bnip3 (Figures 5A, 5B, and S6), the mitochondrial content of cells after Hif1a knockdown was not decreased by myriocin treatment ( Figure 5D). Additionally, Hif1a knockdown hampered the inhibitory effect of myriocin on ROS production and lipid peroxidation induced by erastin or glutamate treatment ( Figures 5E and 5F). Lastly, the protective effect of myriocin against erastin-or glutamate-induced cell death was significantly reduced by Hif1a knockdown Figure 5G). These results strongly indicate that HIF1a is crucial for the cytoprotective effects of myriocin against erastin or glutamate treatment in HT22 cells.

Myriocin treatment stabilizes HIF1a by decreasing its ubiquitination
HIF1a undergoes rapid degradation in normoxia, and its accumulation is controlled by the rates of both synthesis and degradation. Our results demonstrate that myriocin significantly stimulates the protein level of HIF1a with only very minor effect in the level of Hif1a mRNA ( Figures 4A-4C), indicating that myriocin regulates HIF1a level mainly via post-transcriptional events. It has been suggested that the MAPK and PI3K pathways may promote translation of HIF1a ( Figure 6A) (Befani et al., 2012;Masoud and Li, 2015;Mi et al., 2017). We found that inhibiting the MAPK pathway did not interfere with HIF1a expression in HT22 cells ( Figure S7A). On the other hand, when HT22 cells were treated with LY294002, AZD5363, or rapamycin, inhibitors of PI3K, AKT, and mTOR, respectively, HIF1a was significantly decreased even when ll OPEN ACCESS 6 iScience 25, 104533, July 15, 2022 iScience Article cells were co-treated with myriocin ( Figures 6B and S7B). However, myriocin treatment did not increase, but instead decreased the phosphorylation of AKT and P70S6K ( Figures 6C and S7C), indicating that myriocin inhibits the PI3K/AKT/mTOR axis. Therefore, although the level of HIF1a is indeed regulated by PI3K/AKT/ mTOR axis, myriocin did not increase HIF1a by activating this pathway.
Because HIF1a is degraded by proteasome, a proteasome inhibitor MG-132 was added into cell culture with or without myriocin treatment. The co-treatment of both MG-132 and myriocin did not further increase the HIF1a level which was elevated by MG-132 or myriocin alone ( Figures 6D and S7D), indicating that there are some overlapping effects between myriocin and MG132. A cycloheximide chase assay also  iScience Article revealed that HIF1a protein is much more stable in cells pre-treated with myriocin ( Figure S7E). These data indicate that myriocin may stabilize HIF1a by preventing its proteasomal degradation.
To investigate if the ubiquitination of HIF1a is regulated by myriocin, HIF1a protein was immunoprecipitated and its ubiquitination level was analyzed. While myriocin treatment increased the level of the un-ubiquitinated form of HIF1a, ubiquitinated HIF1a was decreased to about 50% ( Figures 6E and S7F). And the addition of DHS counteracted this effect ( Figure S7G). Together, these results suggest that myriocin inhibits the proteasomal degradation of HIF1a by partially preventing its ubiquitination.
Because HIF-1 pathway plays important roles in many physiological and pathological processes in all metazoan organisms, we asked if myriocin also upregulates HIF1a in cells other than HT22. Therefore, the levels of HIF1a in four other cell lines including PC-12 (rat neural cell), HT1080 (human fibrosarcoma cell), GES-1 (human gastric epithelial cell), and SK-Hep-1 (human hepatoma cell) were monitored with or without myriocin treatment. Myriocin treatment increased the protein level of HIF1a in all of these cell types ( Figures 7A and S8A) with no effect on the mRNA levels ( Figure 7B). Additionally, the ubiquitination of HIF1a in PC-12, HT1080, and GES-1 cells was inhibited by myriocin ( Figures 7C and S8B). The baseline ubiquitination of HIF1a in SK-Hep-1 was too low to observe a further decrease ( Figures 7C and S8B). Pre-treatment with myriocin decreased the erastin-induced cell death of PC-12 and HT1080 cells, but it did not promote the survival of GES-1 and SK-Hep-1 cells which had higher basal levels of HIF1a than PC-12, HT1080, and HT22 cells ( Figures 7D and S8C). Therefore, the effect of myriocin on HIF1a is not limited in HT22 although its effects on cell fate are cell-type-dependent.

DISCUSSION
It has been demonstrated that inhibiting de novo synthesis of sphingolipids by myriocin induces stress responses and increases lifespan in yeast cells. This cytoprotective effect of myriocin seems to be conserved in eukaryotes because myriocin treatment also increases lifespan in Caenorhabditis elegans (Cutler et al., 2014). In rodent models, myriocin treatment mitigates age-related diseases including Alzheimer disease, motor neuron degeneration, and Parkinson disease (Geekiyanage et al., 2013;Lin et al., 2019;Petit et al., 2020). The cytoprotective effects of myriocin on yeast cells are established by inducing a state resembling amino acid restriction and the subsequent inhibition of mTOR pathway (Hepowit et al., 2021). However, it is unclear how inhibiting sphingolipid synthesis by myriocin promotes cell survival in metazoan organisms. To facilitate the therapeutic usage of myriocin or myriocin-like compounds, determining the mechanistic basis of the cytoprotective effect induced by myriocin in mammalian cells is critical.
Here, we report that myriocin inhibits ferroptosis induced by glutathione depletion in neuronal HT22 cells. This is so far the first study demonstrating that inhibiting the synthesis of sphingolipids protects cells against ferroptosis. Because ferroptosis induced by oxidative stress in neuronal cells is relevant to aging and age-related neurodegeneration, our work provides a new approach for developing therapeutic or preventive means against those disorders.
Erastin or glutamate treatment in HT22 cells results in glutathione depletion which in turn induces ferroptosis. Glutathione is required for the removal of lipid peroxides by glutathione peroxidase 4 (GPx4), a key enzyme to protect cells from ferroptotic death under oxidative stress. The fact that myriocin treatment iScience Article barely prevents the drop of intracellular levels of glutathione, suggests that the protective effect of myriocin do not rely on the scavenging of lipid peroxide by GPx4. Recent studies have revealed that lipid peroxides can be removed by FSP1 through ubiquinone (CoQ10) or by nitric oxide synthase (iNOS)/ NO independent of GPx4 (Doll et al., 2019;Kapralov et al., 2020), which shares a similar mechanical character as the protective effects of myriocin. However, myriocin pre-treatment significantly suppressed ROS production under glutathione depletion. Additionally, the level of LIP was also decreased, most likely due to the less ROS which leads to the releasement of iron from its storage proteins (Di Tano et al., 2020;Lu and Imlay, 2019;Torti and Torti, 2013). Unlike GPx4 or FSP1, the protective role of myriocin against ferroptosis relies on the prevention of lipid peroxide accumulation caused by the oxidative reaction of polyunsaturated fatty acid with ROS and iron (Boada-Romero et al., 2020;Tang et al., 2019). Therefore, it is likely that the removal of lipid peroxides contributes very little, if any, to the protective effects of myriocin against ferroptosis. To address how myriocin prevents the buildup of oxidative stress induced by glutathione depletion, we performed transcriptome analysis and surprisingly identified the HIF-1 pathway. Although it has been well established that this pathway is involved in cell survival and the inhibition of ferroptosis Poloznikov et al., 2020;Yang et al., 2019), it has never been reported that inhibiting biosynthesis of sphingolipids activates HIF-1 pathway. Additionally, although some studies have demonstrated that HIF-1 pathway or hypoxia plays substantial roles in sphingolipid metabolism (Novgorodov and Gudz, 2011;Testai et al., 2014), if and how sphingolipid metabolism affects HIF-1 pathway is not well established. By validating the activation effect of myriocin on the HIF-1 pathway, this work identifies a novel and interesting connection between sphingolipid metabolism and the HIF-1 pathway. There are many downstream effectors in the HIF-1 pathway which impact many aspects of cell physiology. In this study, we examined PDK1 and BNIP3 because they are key regulators of glycolysis and mitochondrial homeostasis, respectively. Because glycolysis and mitochondrial homeostasis are crucial for energy metabolism and ROS production, their alteration by myriocin is likely to be cytoprotective. (B) Cells treated with myriocin (0.5 mM) for 36 h were subjected to qPCR to measure the relative Hif1a mRNA expression. Error bars represent the mean G SD (n = 3). (C) Ubiquitin/HIF1a co-immunoprecipitation in cells extracts. Cells treated with or without myriocin (0.5 mM) were lysed and immunoprecipitated with anti-HIF1a antibody or IgG as negative control. The immunocomplexes were then immunoblotted using anti-ubiquitin or anti-HIF1a antibody (Output). The levels of HIF1a in cell lysates before immunoprecipitation were also monitored (Input). (D) Cell viability analysis of cells pre-treated with myriocin (0.5 mM) for 36 h before incubating with erastin for 24 h. Error bars represent the mean G SD (n = 3, **p < 0.01, ***p < 0.001).

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iScience 25, 104533, July 15, 2022 11 iScience Article Finally, the regulatory role of myriocin on the level of HIF1a is pinpointed to the proteolysis process because the regulation at transcriptional or translational level has been ruled out. The proteolysis of HIF-1a is initiated by the binding of the von Hippel-Lindau tumor-suppressor protein (VHL), which in turn favors the ubiquitination of HIF1a and targets it to proteasomal degradation (Paltoglou and Roberts, 2007). Although further studies are required to reveal the details about how myriocin alters this process, our results indicate that the ubiquitination of HIF1a is decreased by myriocin treatment which results in the upregulation of HIF1a.
In conclusion, our work demonstrates that inhibiting the de novo synthesis of sphingolipid by myriocin protects neuronal cells against ferroptosis via the HIF-1 pathway. Mechanistically, myriocin increases HIF1a protein by inhibiting its ubiquitination and subsequent proteasomal degradation. Elevated HIF1a in turn stimulates the HIF-1 pathway which promotes cell survival against ferroptosis by rewiring carbon metabolism and promoting redox balance. Future works will aim to understand the cytoprotective roles of myriocin on in vivo models of neurodegeneration to evaluate the potential of myriocin or similar compounds for the prevention or treatment of multiple age-associated neurological disorders.

Limitations of the study
Our data revealed the link between sphingolipid, HIF-1 pathway, and ferroptosis, but the exact underlying mechanism remains to be explored, especially the relationship between sphingolipid synthesis and HIF1a ubiquitination. And in vivo experiments are needed to evaluate the systemic effect of myriocin.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:


STAT3 Role in T-Cell Memory Formation

Along with the clinical success of immuno-oncology drugs and cellular therapies, T-cell biology has attracted considerable attention in the immunology community. Long-term immunity, traditionally analyzed in the context of infection, is increasingly studied in cancer. Many signaling pathways, transcription factors, and metabolic regulators have been shown to participate in the formation of memory T cells. There is increasing evidence that the signal transducer and activator of transcription-3 (STAT3) signaling pathway is crucial for the formation of long-term T-cell immunity capable of efficient recall responses. In this review, we summarize what is currently known about STAT3 role in the context of memory T-cell formation and antitumor immunity.

The primary role of STAT3 is transcriptional regulation, and it preferentially binds GAS elements with the consensus motif TTCN 2-4 GAA [17]. STAT3 target genes are numerous, and genome-wide ChIP-Seq analysis of STAT3 in CD4+ showed about 3000 targets [18]. Moreover, unphosphorylated STAT3 can also act as a transcriptional coregulator with collaboration with other proteins [12,15,19].
STAT3 also has important non-translational activities. It was shown to promote mitochondrial metabolism with an effect on other cellular functions [20][21][22]. During the activation of CD4+ T cells, STAT3 accumulates at mitochondria and sustains high mitochondrial membrane potential skewing differentiation toward the Th17 phenotype [23]. STAT3 is pleiotropic and was shown to play a role in physiology and development [24,25]. Its knockout leads to embryonic lethality [26], and STAT3 signaling is essential for cardiac function and maintaining the differentiation potential of stem cells [27,28]. In the blood compartment, STAT3 is required for the proper functioning of both B cells [29][30][31][32] and myeloid cells [33][34][35]. More importantly for the T-cell context, STAT3 signaling is required for hematopoiesis [36][37][38] and the proper development of the thymus [39][40][41] and can lead to abnormalities if perturbed. Moreover, there is increasing evidence that STAT3 signaling can lead to abnormalities if perturbed. Moreover, there is increasing evidence that STAT3 signaling plays a role at the late stage of T-cell development [42,43]. For that reason, mouse models with a conditional knockout in immature T cells should be interpreted with caution as the observed phenotype in mature T cells can be affected by prior development abnormalities.

STAT3 Role in Th17 and Tfh Differentiation
STAT3 is crucial for the differentiation of specific CD4+ T cells subsets. Th17 is a proinflammatory CD4+ effector subset characterized by IL-17 secretion and its differentiation from naïve CD4+ T cells, which occurs in the presence of TGF-β and IL-6 [44,45]. Through STAT3, IL-6 induces the expression of lineage-defining transcription factor RORγt, and IL-21 and IL-23 receptors. IL-21 and IL-23 then create a positive feedback loop and further increase RORγt. STAT3 also induces IL-17 expression in collaboration with RORγt, and STAT3-targeted deletion impairs Th17 differentiation [46,47]. In the presence of IL-6 and/or IL-21 and the absence of TGF-β during priming, CD4+ T cells can differentiate into Tfh (follicular helper) subset [48]. This T-cell subset is characterized by CXCR5 expression and is necessary for B-cell activation and germinal center formation [49]. BCL-6 is the lineage-defining factor of Tfh, whose expression is also positively regulated by STAT3 [48,50]. It is important to note that, in these settings, lineage-defining factors such as STAT3 promote differentiation into specific CD4+ subtypes, in addition to restricting differentiation into alternative subtypes [51,52]. In support of this idea, Martinez-Fabregas et al. (2019) performed STAT3 ChIP-seq and RNA-seq in human Th1 cells and found that, out of 3400 STAT3 bound genes, only 23 were actually upregulated [53]. Th1 differentiation is driven by the STAT4-T-bet signaling axis and suppresses alternative CD4+ T-cell fates [54].

STAT3 Role in T-Cell Memory Formation
The crucial role of STAT3 in T-cell survival was initially demonstrated by the conditional deletion of this transcription factor in T-cell lineage [55]. In this system, T cells from STAT3-deficient mice were compared with T cells from wild-type mice in vitro. IL-6/STAT3 signaling was shown to have an antiapoptotic effect on T cells independent of Bcl-2 protein. This effect was abrogated in STAT3-deficient cells. IL-6/STAT3 signaling did not have any impact on T cell proliferation. Subsequently, the positive effect of STAT3

STAT3 Role in Th17 and Tfh Differentiation
STAT3 is crucial for the differentiation of specific CD4+ T cells subsets. Th17 is a proinflammatory CD4+ effector subset characterized by IL-17 secretion and its differentiation from naïve CD4+ T cells, which occurs in the presence of TGF-β and IL-6 [44,45]. Through STAT3, IL-6 induces the expression of lineage-defining transcription factor RORγt, and IL-21 and IL-23 receptors. IL-21 and IL-23 then create a positive feedback loop and further increase RORγt. STAT3 also induces IL-17 expression in collaboration with RORγt, and STAT3-targeted deletion impairs Th17 differentiation [46,47]. In the presence of IL-6 and/or IL-21 and the absence of TGF-β during priming, CD4+ T cells can differentiate into Tfh (follicular helper) subset [48]. This T-cell subset is characterized by CXCR5 expression and is necessary for B-cell activation and germinal center formation [49]. BCL-6 is the lineagedefining factor of Tfh, whose expression is also positively regulated by STAT3 [48,50]. It is important to note that, in these settings, lineage-defining factors such as STAT3 promote differentiation into specific CD4+ subtypes, in addition to restricting differentiation into alternative subtypes [51,52]. In support of this idea, Martinez-Fabregas et al. (2019) performed STAT3 ChIP-seq and RNA-seq in human Th1 cells and found that, out of 3400 STAT3 bound genes, only 23 were actually upregulated [53]. Th1 differentiation is driven by the STAT4-T-bet signaling axis and suppresses alternative CD4+ T-cell fates [54].

STAT3 Role in T-Cell Memory Formation
The crucial role of STAT3 in T-cell survival was initially demonstrated by the conditional deletion of this transcription factor in T-cell lineage [55]. In this system, T cells from STAT3-deficient mice were compared with T cells from wild-type mice in vitro. IL-6/STAT3 signaling was shown to have an antiapoptotic effect on T cells independent of Bcl-2 protein.
This effect was abrogated in STAT3-deficient cells. IL-6/STAT3 signaling did not have any impact on T cell proliferation. Subsequently, the positive effect of STAT3 signaling on T-cell survival and its lack of proliferation was confirmed in another model [56]. They vaccinated mice with pigeon cytochrome C with or without concurrent IL-6 administration. IL-6 administration did not lead to more significant primary T-cell division, but instead reduced the apoptosis of antigen-specific T cells and increased their numbers 72 days after immunization. Although recall responses of memory CD4+ from both IL-6 treated and nontreated groups were similar, this study shows the STAT3 role in T-cell memory formation in vivo. The survival benefit of IL-6/STAT3 signaling in CD8+ T cells was demonstrated in the adoptive transfer mouse model [57]. After adoptive transfer, IL6Ra+ CD8+ T cells survived longer and had better recall responses than IL6Ra-counterparts. Durant et al. (2010) performed a ChIP-seq analysis of STAT3 in CD4+ and showed that STAT3 binds to Bcl-2 and activates its expression [18]. Moreover, the addition of STAT5-activating IL-2 could not rescue the viability of STAT3 -/suggesting nonoverlapping roles of STAT3 and STAT5 in CD4+ T-cell survival. Another group used conditional knockout of IL-6 receptor alpha chain in double-positive CD4+ CD8+ T-cell precursors and demonstrated that IL-6/STAT3 signaling is essential for proper CD4+ T-cell memory formation [58]. Wild-type or IL6ra KO mice were immunized with OVA peptide with a second immunization 60 days later. Seven days after the second immunization, CD4+ T cells from IL6ra T-KO hosts were analyzed and showed impaired proliferation and cytokine production in vitro. Interestingly, IL6ra and gp130 are downregulated upon TCR engagement in both CD8+ and CD4+ T cells, demonstrating the importance of IL-6 signaling timing [59]. Supporting previous findings, Oh et al. (2011) showed that STAT3 constrains T-cell proliferation but enhances survival. They used CD4-Cre STAT3 conditional deletion to demonstrate STAT3-driven expression of Bcl-2, FoxO1, and FoxO3a. STAT3 directly bound FoxO1 and FoxO3a promoters and STAT3 deficiency caused downregulation of FoxO target genes: p27Kip1 and IkB. The former (p27Kip1) is a cell-cycle inhibitor, and the latter (IkB) is inhibitor of NF-kB, the pathway that is required for the production of mitogenic IL-2.
The role of STAT3 in driving expression of T-cell memory-related BCL6 and SOCS3 was revealed in the acute LCMV mouse model with conditional knockout of STAT3 in CD8+ T cells driven by granzyme B promoter [60]. IL-21 and IL-10 activate STAT3, and the IL-21/IL-10/STAT3/BCL6/SOCS3 axis was shown to be essential for the formation of memory precursors CD8+ T cells (KLRG1-IL7R+). Removal of any component from this axis led to an impaired accumulation of memory precursors T cells. As a result, significantly fewer memory CD8+ T cells formed in the long term. Mechanistically, SOCS3 reduces IL-12-driven phosphorylation of STAT4, which is known to promote short-lived effector (KLRG1+ IL7R-) and suppress memory precursor differentiation [61]. At least in CD4+ T cells, the SOCS3 locus is directly activated by STAT3 binding [18]. In another study, this group immunized mice with dendritic cells in the presence of different adjuvants [62]. They showed that LPS as adjuvant increased serum levels of IL-6 and IL-10 and resulted in more T-cell memory precursors and better secondary immune response. CpG adjuvant, on the other hand, did not induce the expression of either cytokine and, instead, led to inferior memory T-cell formation. The role of IL-6/STAT3 in memory T-cell formation was also indirectly demonstrated when antigen-specific mouse T cells were cultured with APC in the presence of the cytokine [63]. In this system, IL-6 induced expression of ID3, a transcription factor that is essential for proper differentiation of memory T cells. Interestingly, IL-21, which also activates STAT3 and drives memory formation, did not induce ID3 expression. If anything, IL-21 upregulated expression of ID3-antagonizing transcription factor ID2, likely through STAT4 activation. The role of IL-10 in T-cell memory formation was demonstrated earlier in the Listeria infection model [64]. IL-10 deficient mice had a better primary response but fewer memory T cells and suboptimal secondary response.
ferentiation [63,[65][66][67][68][69]. Depending on the presence, timing, and strength of signals (antigen, costimulatory/inhibitory molecules, cytokines or other signaling molecules, small molecules, ions) that initiate transcriptional reprogramming, T cells can acquire some or all attributes of memory or effector cell lineage [70][71][72][73][74][75][76]. STAT3 signaling is part of the memory formation program and was shown to downregulate T-cell cytotoxic effector functions. Ciucci et al. (2017) cultured CD8+ under Th17 polarizing conditions (IL-6, TGFb, antibodies against IFNγ, IL-4, and IL-12) and showed that T cells expressing wild-type STAT3 repressed cytotoxic gene expression (GZMB, Pfr1, IFNγ, Tbx21, Eomes), whereas STAT3 -/-failed to do so. This effect was mediated, in part, through the STAT3 target gene RORγt [77]. Besides IL-21-IL-10-STAT3-BCL6-SOCS3 axis, there is another mechanistic link between STAT3 and restriction of terminal effector differentiation. STAT3 upregulates expression of FoxO1, which, in turn, represses expression of T-bet, a master regulator of terminal differentiation [78,79]. On the other hand, IL-2/STAT5 signaling is one of the pathways that drive T-cell terminal differentiation and suppress memory formation [68,80,81]. There is evidence that STAT5 and STAT3 act as mutual antagonists. Kalie et al. (2010) demonstrated that on day 3.5 of acute LCMV infection primed T cells can be divided into CD25low and CD25high, which preferentially differentiate into memory precursors (KLRG1-IL7R+) and shortlived effector cells (KLRG1+ IL7R-), respectively [82]. CD25 is the IL-2 receptor alphachain and is required for IL-2 signal transduction [83]. CD25low cells had decreased IL-2/STAT5 signaling but were enriched with the IL-6 signaling pathway that acts through STAT3. In another study, the authors compared murine CD8+ T cell ex vivo expansion in the presence of IL-2, IL-15, or IL-21 [84]. Unlike IL-2 and IL-15, which utilize STAT5 for downstream signaling, IL-21 (STAT3-activating cytokine) increased expression of memory-related genes (Lef1, Sell, Tcf7) and repressed expression of effector ones (Eomes, GZMB, IL2Ra). Tcf7 is a direct target of STAT3 and is vital for memory formation [18,66]. Moreover, IL-21 reduces the expression of IL-2-driven Eomes when both cytokines are present simultaneously. Compared with IL-2, IL-21-treated T cells showed superior antitumor activity in the B16 melanoma subcutaneous model. In the acute LCMV model, IL-21 deficiency led to lower numbers of IL-2 producing CD8+ T cells [85]. IL-2 production is a feature of naïve and memory T cells and is repressed in effector cells in a BLIMP1- On the other hand, IL-2/STAT5 signaling is one of the pathways that drive T-cell terminal differentiation and suppress memory formation [68,80,81]. There is evidence that STAT5 and STAT3 act as mutual antagonists. Kalie et al. (2010) demonstrated that on day 3.5 of acute LCMV infection primed T cells can be divided into CD25low and CD25high, which preferentially differentiate into memory precursors (KLRG1-IL7R+) and short-lived effector cells (KLRG1+ IL7R-), respectively [82]. CD25 is the IL-2 receptor alpha-chain and is required for IL-2 signal transduction [83]. CD25low cells had decreased IL-2/STAT5 signaling but were enriched with the IL-6 signaling pathway that acts through STAT3. In another study, the authors compared murine CD8+ T cell ex vivo expansion in the presence of IL-2, IL-15, or IL-21 [84]. Unlike IL-2 and IL-15, which utilize STAT5 for downstream signaling, IL-21 (STAT3-activating cytokine) increased expression of memory-related genes (Lef1, Sell, Tcf7) and repressed expression of effector ones (Eomes, GZMB, IL2Ra). Tcf7 is a direct target of STAT3 and is vital for memory formation [18,66]. Moreover, IL-21 reduces the expression of IL-2-driven Eomes when both cytokines are present simultaneously. Compared with IL-2, IL-21-treated T cells showed superior antitumor activity in the B16 melanoma subcutaneous model. In the acute LCMV model, IL-21 deficiency led to lower numbers of IL-2 producing CD8+ T cells [85]. IL-2 production is a feature of naïve and memory T cells and is repressed in effector cells in a BLIMP1-dependent manner [86]. Interestingly, IL-21 can also induce BLIMP1 expression through STAT3 activation [87]. The authors suggested that an IL-21/BCL6/BLIMP1 incoherent feed-forward loop can explain this discrepancy. IL-21 drives the expression of both BCL6 and BLIMP1 but BCL6 represses BLIMP1 and tunes the balance between these two transcriptional factors.
These studies, however, did not address the molecular mechanism of STAT3-STAT5 antagonism. In the Th17 differentiation setting, this mechanism was studied in greater detail. STAT5 was shown to restrict STAT3-driven RORγt expression and to compete with STAT3 DNA binding at IL-17 locus in CD4+ T cells [88,89]. STAT5 recruitment to the IL-17 locus was associated with decreased H3K4 acetylation and increased binding of NCoR2 corepressor. Although in the Th17 differentiation setting, these studies demonstrate that STAT5 can directly affect STAT3-driven gene expression, one can speculate that STAT5 may have the same direct effect on STAT3-driven genes necessary for memory formation. This idea is supported by a study indicating that IL-2 signaling suppressed BCL6 expression in Th1 cells [90]. The authors showed that STAT5 activated by IL-2 directly bound and repressed BCL6 expression. STAT3 was also shown to share almost 60% of binding sites with STAT5 in murine CD4+ cells [91].
Although the above evidence clearly argues for mutual antagonism, STAT3 and STAT5 can act cooperatively. For example, at least at low doses of IL-2, optimal CD25 (IL2Ra) expression on activated T cells requires intact STAT3 [92]. IL-21-activated STAT3 can positively regulate IL2Ra expression through super-enhancer binding [91]. This is unexpected, given the opposite roles of IL-2 and STAT3 signaling in memory formation.

Role of STAT3 Signaling in T-Cell Antitumor Immunity
Although the role of STAT3 in memory formation seems to be prominent, its translation into antitumor settings is less clear. There is no consensus on the role of STAT3 in antitumor immunity. Depending on the model, STAT3 activation was either beneficial or detrimental to the ability to fight the tumor. By reviewing studies on STAT3 in cancer settings, we attempt to relate the STAT3 role in memory formation to antitumor immunity. In an early study of STAT3, the Mx1-Cre-loxP system was used to knockout STAT3 in hematopoietic cells in adult mice [93]. STAT3 -/hosts rejected melanoma (B16), and bladder carcinoma (MB49), and these rejections were dependent on the T cells' presence. STAT3 -/hosts had increased IFNγ production by CD8+ T cells, tumor infiltration, and decreased number of Tregs. This group demonstrated a similar antitumor effect using STAT3 conditional deletion in the T-cell lineage (CD4-Cre) and adoptive transfer model [94]. They also observed increased proliferation of STAT3 -/-T cells. In a subsequent study, the same group elucidated the mechanism of STAT3 -/increased antitumor immunity [95]. Using the same STAT3 conditional deletion in the T-cell lineage, they demonstrated that STAT3 -/-CD8+ T cells had increased IFNγ production and increased CXCR3 expression in subcutaneous melanoma (B16) and lung carcinoma (3LL) models. IFNγ promoted CXCL10 secretion by myeloid cells in the tumor and, as a result, attracted more CXCR3 expressing effector T cells. The negative effect of STAT3 signaling on CXCR3 expression was confirmed by another study [96]. The authors showed that increased IL17A in serum from colorectal cancer patients activated STAT3, leading to the downregulation of CXCR3 in CD8+ T cells and reduced tumor infiltration. High IL17A levels also correlated with worse patients' survival. Similar to Yue et al. (2015), induction of cytokine secretion was observed in the glioma model after T-cell transfection with miR-124 [97]. MiR-124 downregulated STAT3 in T cells with a concomitant increase in IL-2, IFNγ, and TNF-α production. However, in another study, miR-124 had an opposite effect on STAT3 signaling [98]. The authors showed that the MeCP2-miR-124 axis suppresses SOCS5 expression in CD4+ T cells. SOCS5 inhibits STAT1 and STAT3 activation, thereby preventing Th1 and Th17 differentiation. The apparent contradiction in the miR-124 role can be attributed to its various effects in CD8+ and CD4+ T cells. Alternatively, STAT1 and STAT3 may have different sensitivities with respect to SOCS5.
In a recent study, the connection between tumor progression and STAT3 driven metabolism was discovered [99]. They showed that in a spontaneous breast cancer model, ablating leptin-driven STAT3 signaling in CD8+ T cells enhanced the antitumor effect by reducing fatty acid oxidation (FAO) and promoting glycolysis. Glycolysis is essential for effector T-cell expansion and synthesis of effector molecules [100][101][102]. Interestingly, they also showed that PD1 signaling, known to induce FAO and inhibit glycolysis, requires intact STAT3 [103]. However, it is not yet clear whether such a metabolic shift restricts antitumor immunity. Published studies demonstrate that it can be beneficial [104,105]. In a recent study, subdermal melanoma (CL24) and pancreatic adenocarcinoma (PanCO2) models were treated with leptin-expressing oncolytic vaccinia virus [106]. Leptin-STAT3 signaling increased mitochondrial content, IFNγ, and TNFa production by CD8+ T cells.
As a result, only leptin-engineered viruses improved hosts' survival. Although confounded by potential leptin effects on tumor cells, this study demonstrates the positive role of T-cell STAT3 signaling in antitumor immunity. As illustrated above, STAT3 signaling restricts glycolysis and promotes FAO in T cells. A similar metabolic shift is also observed during memory formation and was also shown to improve antitumor immunity [107,108].
The role of STAT3 in the antitumor activity of the CD4+ compartment is even more difficult to dissect. Besides memory formation, STAT3 signaling promotes differentiation of naïve CD4+ into Th17 and Tfh. These subsets can have both protumor and antitumor activity depending on the model [109][110][111]. For example, in one study, breast cancer patients had reduced IL6-driven STAT3 phosphorylation in CD4+ T cells. This correlated with a worse survival prognosis and was associated with impaired Th17 differentiation suggesting a decisive role of STAT3 activation [112]. In another mouse model study, IL-6/STAT3 signaling impaired CD4+ T-cell differentiation into the Th1 subset and compromised antitumor immunity [112]. Interestingly, Th17 cells were shown to exhibit prolonged longevity when compared with the Th1 subset [113,114]. Th17 cells also preserved differentiation plasticity and gave rise to Th1-like progeny that exerted antitumor activity. This suggests that Th17 cells are more stem-like than other Th subtypes. From these studies, it appears that Th17 differentiation is interconnected with memory formation potential. Another study showed that the gene expression signature of Tfh is close to the signature of CD8 T-cell memory precursors [115]. Unlike short-lived Th1 cells, Tfh cells were able to become memory cells and (similarly to CD8+ memory precursors) expressed higher levels of BCL6, TCF7, and ID3 and lower levels of IL2Ra and ID2. Thus, additional studies are required to elucidate the pleiotropic role of STAT3 in CD4+ T-cell subsets.
It is useful to look at STAT3 signaling in T cells in the context of a broader question about the role of memory T cells in antitumor immunity. Although not indisputable [116], there are reasons to believe that less differentiated T-cell subsets have superior antitumor efficacy when compared with their more differentiated counterparts [117][118][119][120][121][122][123][124]. Indeed, when preinfusion CAR T cells from 41 chronic lymphocytic leukemia patients were analyzed, CAR T cells from complete responders and partial responders were enriched for memory precursor effector cell (MPEC) genes and IL-6-STAT3 pathway [117]. CAR T cells with favorable CD27+PD1-had increased levels of gp130 expression and STAT3 phosphorylation, indicating the active role, as opposed to the mere biomarker role, of this pathway. It is noteworthy that the IL-6 signaling pathway was also enriched in CD25low effector cells in the LCMV model [82]. These cells were more likely to differentiate into memory precursors as opposed to short-lived effector cells. In a leukemia mouse model, CAR constructs incorporating both STAT5 and STAT3 binding motifs were engineered [125]. It was shown to activate both STAT5 and STAT3 and improve antitumor efficacy in leukemia (NALM6) mouse models. Although in this model, the antitumor effect of STAT3 signaling cannot be separated from STAT5, in vitro stimulated CAR T cells without STAT3 motif had decreased production of IL-2 and a lower number of less differentiated CD45RA+CD62L+CCR7+ CAR T cells. In the subcutaneous mesothelioma (M108) model, CAR T cells expanded in Th17-polarizing conditions provided superior tumor control [126]. A beneficial effect of human T-cell activation in the presence of IL-21 was shown also revealed [127]. In this study, CAR T cells targeting CD19+ were expanded in the presence of only IL-2 or in the presence of IL-2 and IL-21. In the latter case, CAR T cells showed better in vitro proliferation and a significant reduction in the leukemia NALM6 burden (NSG model). Consistent with Hinrichs et al. (2008), IL-21 treatment reduced Eomes expression in vitro. However, as opposed to Hinrichs et al. (2008), it also increased granzyme B expression, highlighting the model differences.
The benefit of less differentiated (memory) T cells for antitumor immunity lies partly in their ability to expand upon antigen encounter significantly and partly in their longterm survival advantage. Some of this survival advantage can be attributed to metabolic flexibility that can be crucial in the hostile tumor microenvironment. STAT3 both drives proper memory differentiation at early stages and modulates T-cell metabolism at the tumor site, contributing to antitumor response. Both aspects lead to better T-cell survival and persistence. On the other hand, memory T cells may have restricted cytotoxic and migration capacity, and in the short term, more differentiated T cells can have an antitumor advantage. However, it is also possible that mouse models are too short of capturing all the dynamics of antitumor response driven by memory T cells and observed in patients. In terms of metabolism, for hypoglycemic tumors, FAO induction seems to be beneficial, whereas, in an environment with sufficient nutrients, it will restrict T-cell expansion by inhibiting glycolysis. Hence, in cellular therapy, it may be reasonable to mimic physiological immune response and combine highly differentiated effector cells with less differentiated memory cells. The former will cause initial tumor reduction and prevent overstimulation of coadministered memory cells. The latter will provide antitumor effects after effector cells contract.

STAT3 Mutations Linked to T-Cell Memory
Rare Mendelian disorders or naturally occurring mutations in the tumor can provide much information about the physiological role of proteins harboring them. Such mutations can be correlated to their manifestation in cellular phenotype within the physiological environment.
Direct evidence of a role for STAT3 in human T-cell memory formation emerged from a study of individuals with the autosomal-dominant hyper-IgE syndrome (AD-HIES), which is caused by dominant-negative STAT3 mutations [128,129]. Individuals with AD-HIES, among other abnormalities, are susceptible to viral and bacterial infections. Importantly, in such individuals, fewer memory T cells (CD45RO+ CD27+) form, and their naïve T cells have impaired expression of BCL6 and SOCS3, which contribute to memory formation [60,[130][131][132]. In line with effector-memory antagonism, increased expression of granzyme B in CD8+ TEMRA and EM subsets from AD-HIES individuals was also demonstrated [129].
Although STAT3 dysregulation often occurs in many cancer types, the role of STAT3 in large granular lymphocytic (LGL) leukemia is of particular interest [133,134]. LGL leukemia is a rare chronic lymphoproliferative disorder of mature T (CD4+, CD8+, or gamma/delta) or NK cells [135,136]. It is an indolent disease with two-thirds of symptomatic cases having splenomegaly, anemia, or neutropenia [137,138]. The origin of the disease is unknown, and the current T-cell LGL leukemia model suggests a two-step mechanism [139][140][141]. First, unknown antigen drives oligoclonal T-cell expansion. Then, dysregulation of survival pathways and, potentially persistent antigen, allow specific clones to avoid T-cell contraction and establish clonal dominance. Importantly, STAT3 seems to play a central role in this survival benefit and was shown to be activated in LGL cells, both by mutations and extrinsic factors, and its inhibition led to apoptosis of T-LGL cells in vitro [142][143][144]. STAT3 mutations appear in up to 70% of T-LGL leukemia cases. Mutations in the SH2 domain are most prevalent, with Y640F being the most common substitution. Y640F, D661V, among other mutations, make STAT3 more prone to dimerization, leading to increased transcriptional activity [145]. On the other hand, T-LGL leukemia cases without STAT3 mutations are still associated with increased STAT3 signaling. High amounts of IL-6 were observed in LGL leukemia individuals and provided antiapoptotic benefits through STAT3 activation [146]. Similarly, the downregulation of SOCS3, capable of reducing STAT3 signaling, was also observed in patients. PTPRT is a phosphatase responsible for the dephosphorylation of Tyr705 on STAT3 with subsequent STAT3 deactivation [147]. Consequently, PTPRT V995M mutation in LGL patients was also associated with increased STAT3 activity ). BCL11B H126R was associated with increased STAT3 signaling, although there is no known connection between BCL11B and STAT3. Interestingly, in STAT5 Y665F-mutated cases, STAT3 signaling was also activated. This STAT5 mutation is an activating one, suggesting positive crosstalk between STAT3 and STAT5, in contrast to the negative one mentioned in the context of Th17 differentiation [148].
LGL patients with STAT3 mutations are at higher risk of developing rheumatoid arthritis [149]. Interestingly, in a mouse model (adoptive transfer of bone marrow that was transduced with STAT3 mutations) activating STAT3 (Y640F or D661V) mutations by themselves could not induce LGL disease [150]. All in all, the main lesson to learn from TGL leukemia is that STAT3 signaling is associated with a significant survival advantage for T cells, even in the absence of other oncogenic mutations. Such survival is similar to cell survival in memory T cells, which can persist for a very long time after antigen encounter.
STAT3 was reported to be constitutively activated in the late-stage cutaneous T-cell lymphoma (CTCL) [151]. By transfecting tumor cells with dominant-negative STAT3, the authors showed that blocking STAT3 signaling leads to apoptosis. Interestingly, IL-21 expression is also associated with disease progression and with increased mortality in CTCL [152]. It is tempting to speculate that, in this setting, IL-21 activates STAT3 leading to survival benefit.
Mutations in STAT3 are also implicated in the development of autoimmune diseases, including type 1 diabetes [153]. Recently, a mouse model heterozygous for STAT3 K392R gain-of-function mutation was generated [154]. The authors demonstrated that STAT3 K392R mutation in CD8+ T cells counteracts exhaustion, increases cytotoxicity, and can accelerate the development of T1D.

STAT3 Signaling in the Context of IL-6 Blockade
Cytokine release syndrome (CRS) is a common complication after CAR T product infusion and is accompanied by an extensive release of IL-6 [155]. Now it is successfully treated with tocilizumab (anti-IL-6R antibody) [156]. There is no evidence either from CAR T clinical trials (Kymriah, Yescarta, CD22-BBz CAR T) or from other T-cell-engaging therapies (CD123/CD3 DART or CD19/CD3 BiTE antibodies) that IL-6 blockade has a negative impact on T-cell functionality and antitumor response [157][158][159][160][161][162]. The evidence that IL-6 blockade does not affect therapeutic response implies that at least shortly after infusion, IL-6-STAT3 signaling is dispensable for CAR T cells. This discrepancy is likely explained by the fact that at the end of ex vivo expansion CAR T cells differentiate and lose the expression of the gp130 receptor. Supporting this, Fraietta et al. (2018) showed that only the CD27+PD1-population (least differentiated subset) of CAR T cells expresses gp130 and activates STAT3 upon IL-6 administration [117]. Moreover, gp130 expression is largely restricted to naïve and central memory T-cell subsets from peripheral blood and is coexpressed with CCR7 (unpublished data). However, the presence of soluble gp130 and IL-6Ra receptors in patients after CAR-T infusion raises the possibility that T cells without gp130 expression can still respond to IL-6 [163]. Future studies are needed to further elucidate the role of IL-6 blockade on T cells.

Conclusions
Considering all the above, there is increasing evidence that STAT3 is required for T-cell memory formation. However, this is largely based on studies that used conditional knockouts in mouse T-cell precursors.
There is a need for cleaner models that interrogate STAT3 in mature T cells. They will eliminate potential effects of perturbed STAT3 that manifested at late stages of T-cell development. One option is CD8a-Cre system (Cre expression driven by the core E8I enhancer and Cd8a promoter), which deletes a gene in mature CD8+ but not CD4+ T cells [164]. GZMB-Cre system can also be used (Cui et al. 2011). Alternatively, one can consider CRISPR-Cas9-mediated gene editing in mature T cells, a system that has been validated in both human and mouse T cells [165][166][167].
STAT3 signaling in human mature T cells is still understudied and most of the evidence is still correlative. Although some parallels between human and mouse models can be drawn, no systematic effort of comparing STAT3 signaling in human and mouse T cells has been made.
In terms of antitumor immunity, STAT3 signaling in T cells seems to be highly contextdependent. There is evidence of both positive and negative roles of this signaling axis in T-cell antitumor immunity. Its negative role can be attributed to STAT3 inhibition of CXCR3-driven tumor trafficking. Hence, inhibition of STAT3 signaling in T cells may be desirable for tumors expressing CXCR3 ligands (CXCL9, CXCL10, CXCL11). Nevertheless, for tumors that do not express these chemokines or blood cancers, STAT3 activation in T cells might provide a persistence advantage with no reduction in trafficking. With STAT3 signaling promoting FAO and restricting glycolysis, STAT3 activation might be beneficial for low glycose environments, where alternative metabolic routes are required for T-cell survival and expansion. On the other hand, if glycose is abundant, STAT3 inhibition can increase T-cell expansion, at least in the short term.